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An LC-MS/MS Clinical Research Method for the Measurement of 25-OH Vitamin D2 and D3 Metabolites

Summary In many clinical research laboratories, the sensitive and reproducible measurement of vitamin D metabolites can be a challenge. Older methods involving the use of immunoassay or other technologies are unable to provide the level of information on these important vitamin D metabolites that many researchers are seeking to measure. W hile LC-MS is capable of making the specific and analytically sensitive measurement of these vitamin D metabolites, preparing samples adequately and efficiently has always been an issue that has not been easy to overcome. T he method described here takes best advantage of the automated sample preparation capabilities of the ACQUIT Y UPLC OSM in the analysis of vitamin D metabolites. T his highly automated method will reduce both the direct staff and instrument time required to perform these analyses when compared to other LLE or offline analysis techniques, and allow for rapid, simultaneous analysis of these vitamin D metabolites. T he C8 sorbent used for extraction and the ACQUIT Y UPLC CSH Hexyl-P henyl Column used in this study also showed excellent performance in terms of sample clean-up and separation of these molecules (Figure 2). Furthermore, both the intra- and inter-assay variability of the method were assessed to be excellent (Tables 1 and 2). Even at low nmol / L concentrations of either vitamin D metabolite, the calculated CVs were typically less than 5%. T he robustness of the method makes it a good option for routine measurement of vitamin D metabolites in a complex matrix such as plasma. In this study, a rapid clinical research method using the ACQUIT Y UPLC OSM for the measurement of vitamin D metabolites has been developed. T he method is suitable for routine use and the small volume of serum used (150 µL) is desirable for clinical research laboratories that are sample limited.
Category 2. Symbiosis Application Notes
These are application notes that have been made in our application lab. They were based on customer demo's and customer method development requests. They include sorbent screening, a calibration curve and are optimized for carry over. They are not always (completely) validated
Matrix plasma
Market OSM Clinical
Cartridge Waters MassTrak C8 Online SPE Cartridges
Detection Waters Xevo® TQ-S MS
Compounds 25-OH D2 and 25-OH D3, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 vitamin D
Compound Group Vitamin D, Vitamin
Contact Martijn van Faassen, Ido Kema, and Kendon Graham
Account University Medical Center Groningen, Groningen, The Netherlands; Waters Corporation, Milford, MA, USA
Published poster, Technical Brief
Date 4-8-2015
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Remarks waters P/N: 720005345EN

Status published